DISEASES OF AQUATIC ORGANISMS Vol. 46: 79-82,2001 Published August 22 Dis Aquat Org

NOTE

First published record of the pathogenic monogenean parasite melleni () from Australia

Marty R. Deveney, Leslie A. Chisholm, Ian D. Whittington"

Department of Microbiology and Parasitology, School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia

ABSTRACT: The monogenean Neobenedenia melleni (Mac­ families from 3 orders as wild hosts of N. melleni. The Callum, 1927) Yamaguti 1963 is a well-known and virulent shape and size of the body and haptoral sclerites and pathogen in culture conditions recorded from the skin of the shape of the testes are important taxonomic charac­ many worldwide. Until now, N. melleni has not been reported from wild or cultured fish in Australian ters that vary considerably in N. melleni (see Whitting­ waters. This study documents a recent outbreak of N. me11eni ton & Horton 1996). which has led to significant prob­ that occurred on Lates calcarifer (barramundi) cultivated in lems in defining the taxon. Taxonomic difficulties sea cages in Hinchinbrook Channel between Hinchinbrook remain because Ogawa et al. (1995) recognised N. Island and mainland Queensland, Australia, which resulted in the loss of 200000 fish (50 tonnes). The origin of this outbreak girellae as a species distinct from N. melleni (see also is unclear because N. melleni has not been recorded from any Ogawa & Yokoyama 1998, Koesharyani et al. 1999) wild host species in Australia and strict quarantine reg­ whereas Whittington & Horton (1996) synonymise N. ulations exclude the possibility of its introduction on imported girellae with N. melleni. The definition of N. melleni is fish. We propose that N. melleni occurs naturally on wild the subject of ongoing debate. We follow the decision of populations of some teleost species in Australian waters and that the few surveys of wild fish conducted along the east Whittington & Horton (1996). Throughout this paper, N. coast have failed to report this species. The possibility that un­ melleni is defined sensu Whittington & Horton (1996). characteristically low water temperatures led to the outbreak The above characteristics make Neobenedenia mel­ is discussed. leni a monogenean species of considerable interest. KEY WORDS: · Capsalidae . Neobenedenia Knowledge of its global distribution is particularly sig­ melleni . Sea cage aquaculture . Lates calcarifer . nificant because of its pathogenicity. N. melleni was Ectoparasitic disease· Australia described from numerous fish species in the New York Resale orrepublication notpermitted _ Aquarium (MacCallum 1927) and its tTUe origin (Le., without written consent of the publisher the fish species on which it was introduced) has long been debated, but remains unknown (Whittington & The capsalid monogenean Neobenedenia melleni Horton 1996 and references therein). There are pub­ (MacCallum, 1927) Yamaguti 1963 is an infamous lished records of N. melleni from the Caribbean Sea, pathogen of fish for several reasons. N. melleni is the West Atlantic Ocean, the east and mid-Pacific recognised as a lethal pathogen of captive marine Ocean, and the Red Sea (Whittington & Horton 1996). , whether held in aquaria (e.g., MacCallum Ogawa et al. (1995) reported N. girellae from 14 spe­ 1927, Jahn & Kuhn 1932, Thoney & Hargis 1991) or in cies (6 families; 3 orders) of cultured marine in sea cages for aquaculture (e.g., Kaneko et al. 1988, Japan and provided strong evidence for continuous Mueller et al. 1992). Furthermore, unlike most monoge­ introduction of the pathogen on the unregulated nean parasites, N. melleni is notorious for its lack of importation of amberjack fry (Seriola dumerili Risso) host-specificity. Whittington & Horton (1996) noted from Hong Kong and Hainan, China. Koesharyani et records from more than 100 captive and wild teleost al. (1999) reported N. girellae (likely to be N. melleni species in more than 30 families from 5 orders. Bullard sensu Whittington & Horton [1996). but no voucher et al. (2000) list 27 teleost species from 18 genera in 14 material was deposited) from serranids in a research station in Bali. The list of wild hosts for N. melleni by •Corresponding author. Bullard et al. (2000) confirms the Caribbean area as a E-mail: [email protected] focus for this monogenean species, adding Puerto Rico,

© Inter-Research 2001 80 Dis Aquat Org 46: 79-82, 2001

Grand Cayman Island and the Gulf of Mexico to the in Canada balsam beneath a coverslip on glass slides. growing list of localities. Some unstained individuals were mounted directly in Restricted surveys of wild marine teleosts by Whit­ Hoyer's medium (Pritchard & Kruse 1982) to permit tington & Horton (1996) failed to find Neobenedenia close examination of heavily flattened specimens to melleni at the northern and southern tips of the Great ensure that haptoral sclerites lay as flat as possible to Barrier Reef in Australia, but only small sample sizes of reveal their profile (as recommended by Whittington & a limited range of host species were examined. Whit­ Horton 1996). All specimens were measured using a tington & Horton (1996) commented that it was impor­ computerised digitising system similar to that de­ tant to determine whether Neobenedenia spp., and N. scribed by Roft & Hopcroft (1986). All measurements melleni in particular, occurred in Australian waters are quoted in micrometres as the range followed by the because of the potential threat posed to fin-fish mari­ mean in parentheses. Terminology follows Whittington culture as shown in other countries. In August 2000, & Horton (1996) and discussed further by Whittington personnel at a marine barramundi farm in Queensland et al. (2001). contacted lOW about a 'fluke problem' on cultivated Results. All 4 barramundi specimens examined were stocks of Lates calcarifer (Bloch). Here, we report N. infected with capsalid monogeneans. Live worms were melleni for the first time from Australia. recovered from the fins, 'skin' (= body flanks), head Materials and methods. The monogenean problem and eyes. Infection intensities ranged from 12 to over was first reported to IDW on 16 August 2000. Culture 400 specimens on the most heavily infected barra­ conditions for the barramundi on which the outbreak mundi (total length 145 mm). No capsalids were found was reported were as follows. Specimens of Lates on the gills or gill arches. calcarifer up to 150 mm in total length were main­ Monogeneans from each barramundi examined tained in 2 different kinds of polyethylene mesh cages: matched the redescription of Neobenedenia melleni 2 up to 50 'nursery' cages (1 m , 3 m deep; mesh size by Whittington & Horton (1996). Shapes of haptoral 12 mm) or 6 'grow-out' cages (10 x 5 m, 3 m deep; mesh sclerites (Fig. 1), eggs and measurements of capsalids size 20 mm). Larger fish were maintained in 2 cages removed from L. calcarifer (Table 1) are similar to 10 x 10 m, 3 m deep, or in 5 cages 26 x 10 m, 3 m deep, those provided by Whittington & Horton (1996) for N. each with a mesh size of 25 mm. All cages were located melleni. It should be noted, however, that the low host in the Hinchinbrook Channel, between Hinchinbrook specificity of N. melleni and likely associated 'host­ Island and mainland Australia (18 0 29' 00" S, induced morphological variation' (Whittington & Hor­ 1460 16' 00" E). Additional predator nets were de­ ton 1996), a phenomenon that remains unquantified, ployed with mesh sizes of 50 or 100 mm. Hinchinbrook appears to result in broad size ranges for this taxon. We Channel varies in depth according to tide, but water conclude that the outbreak on these cultivated barra­ depth is approximately 4.5 m on an average tide across mundi in sea cages in Queensland was N. melleni. the farm lease. Stocking density varied, but was esti­ 3 mated at 25 kg of fishm- . Four specimens of Lates calcarifer (total length range 128 to 174 mm) were air freighted from Cairns to Bris­ a bane in oxygenated seawater. Before examination, each fish was killed by pithing and dorsal chordotomy, placed in a Petri dish and immersed in seawater fil­ tered through 2 sheets of Whatman No. 1 filter paper. Each fish was examined for parasites using a stereodis­ secting microscope with incident illumination. Live monogeneans were transferred to small Petri dishes containing filtered seawater where many specimens laid eggs. Numerous adult specimens were flattened beneath slight coverslip pressure and preserved in 10 % buffered neutral formalin. Other specimens were killed instantly in near-boiling seawater and preserved unflattened in 10% buffered neutral formalin. Some flattened and unflattened material was left unstained, Fig. 1. Haptoral sclerites from the monogenean Neobene­ but other specimens were stained using Semichon's denia melleni sensu Whittington & Horton (1996) collected aceto-carmine or Mayer's acid haemalum (Humason from cultivated Lates calcariferin northern Queensland, Aus­ tralia. (al Accessory sclerite; (bl anterior hamulus; (cl posterior 1979). Specimens were dehydrated in a graded hamulus. Drawn from fresh specimens prepared in Hoyer's ethanol series, cleared in cedar wood oil and mounted medium. Scale bar = 125 pm Deveney et al.: First report of Neobenedenia melleni from Australia 81

Table 1. .Measurements of specimens of Neobenedenia There are parallels between the sudden outbreaks melleni sensu Whittington & Horton (1996) from cultivated of Neobenedenia melleni on cultured Oreochromis Lates calcarifer in northern Queensland, Australia. Haptoral mossambicus (Trewavas) (tilapia) in Hawaii (Kaneko sclerites were measured from 10 heavily flattened, mounted specimens (vouchers in the Queensland Museum [QM]: et al. 1988). on cultured amberjack in Japan (Ogawa et G218291-300); soft body parts were measured from 10 unflat­ al. 1995) and on barramundi cultured in Hinchinbrook tened, mounted specimens (vouchers in QM:G218281-90) Channel in Australia (present study). Kaneko et al. (1988) noted that the natural host of N. melleni in Parameter measured ()lm) Range (mean) Hawaiian waters had not been identified despite an extensive survey of Hawaiian marine fishes for mono­ Total length 2230-4190 (2890) geneans conducted previously by Yamaguti (1968). Maximum width 997-1560 (1290) length 667-903 (809) Kaneko et al. (1988) therefore suggested that N. Haptor width 663-916 (793) melleni may have been introduced into Hawaii after Accessory sclerite length 131-190 (165) Yamaguti's (1968) survey. Ogawa et al. (1995) pro­ Anterior hamulus length 198-390 (252) vided strong evidence for continuous introduction of Posterior hamulus length 91-163 (130) N. melleni (identified as N. girellae) into Japan by the Anterior attachment organ diameter 191-374 (312) Pharynx length 191-374 (312) unregulated import of amberjack fry, Senoia dumeriii, Pharynx length 275-335 (293) from Hong Kong and Hainan, China. The same 2 sce­ narios are possible for the outbreak that we report in Australia: either the pathogen occurs naturally on wild There was evidence of loss of scales, some epidermal specimens of 1 or more marine teleost species near the damage, lifting and loosened scales, and epidermal farm where the outbreak occurred or the capsalid was lesions on the 2 most heavily infected Lates caicarifer introduced on imported fish. The latter possibility is specimens. The remaining 2 specimens had fewer than unlikely because Australia has strict regulations on the 25 worms each and appeared relatively healthy with importation of all organisms from overseas and author­ few external signs of pathology. Estimates of the total ities maintain that there is no chance that any marine barramundi losses resulting from infection by N. fish species was imported and stocked locally. The melleni were 200000 fish (approximately 50 tonnes) most likely origin of N. melleni in Australia, therefore, with a value of about AUS $500000 (= US$277 000). is a wild population of 1 or, perhaps, several fish spe­ Twenty voucher specimens of Neobenedenia mei­ cies. The current report adds to the mystery about the ieni collected in this study are deposited in The natural source of N. melleni, which has baffled para­ Queensland Museum, PO Box 3300, South Brisbane, sitologists since its description from the New York Queensland 4101, Australia (Accession G218281-300). Aquarium. Discussion. This is the first confirmed report of Neo­ If Neobenedenia melleni has been present in eastern benedenia melleni from Australia, but sources at the Australian waters on a wild teleost host (or hosts). why farm where the outbreak arose and local professionals did it suddenly transfer to cultivated Lates calcarifer associated with the aquaculture industry nearby sug­ and reach epidemic proportions so quickly? We pro­ gest that N. melleni infections have occurred there pose that a 3 wk period of low (19°C) seawater before, although not on the scale observed in August temperature in late July to early August 2000, unusual 2000. Previous outbreaks of N. melleni may have been for this region, precipitated the outbreak. A low, or a misdiagnosed as Streptococcus infection presumably sudden drop in, temperature is known to stress barra­ because wounds caused by the monogeneans may mundi (Rimmer 1995, Anderson 1996). Disease, espe­ have become secondarily infected by bacteria. cially among barramundi fingerlings, is more common The present report of Neobenedenia melleni from in winter (Rimmer 1995) and low temperatures slow the east coast of Australia extends the Pacific Ocean the production of antibodies and phagocytes and distribution of this pathogen (Whittington & Horton reduce the capacity for barramundi to heal wounds 1996). Our report is also a new confirmed host record, (Anderson 1996). We argue that natural infections of N. although Leong (1997) listed 2 capsalid species (Bene­ melleni probably occur on 1 or more wild fish species denia sp. I and Neobenedenia sp. II) from cultivated around the sea cages where the barramundi are culti­ Lates caicarifer in southeast Asia. Further studies of vated. At seawater temperatures above 20°C, the these capsalid species from various cultivated marine barramundi can likely combat N. melleni infections fin-fishes in southeast Asia are required because host because their immune system is working optimally. distribution data (Whittington et al. 2001) suggest that However, in late July and early August 2000, when the Neobenedenia sp. II may be N. melleni. If this is the seawater temperature fell to 19°C, the immune sys­ case, then N. melleni is widespread throughout Pacific tems of the cultivated L. caicarifer were likely com­ waters. promised. This, combined with the high fish density in 82 Dis Aquat Qrg 46: 79-82, 2001

the sea cages, resulted in the N. Jl1ell€ni.epidemic. This ture Centre, Walkamin, p 29-31 scenario may also explainWI:J.~t.l:J.e outbreak by N. Bullard SA, Benz GW, Overstreet RM, Williams EH Jr, Hem­ melleni on barramundi subsided so rapidly. During dal J (2000) Six new host records and an updated list of wild hosts for Neobenedenia melleni (MacCallum) (Mono­ seawat~~t~:qJ.pe:rai:ure September 2000, the rose above genea: Capsalidae). Comp Parasito167:190-196 20°C and the effeCtiv:~ess.'o,fJbe ij;p:lpmne system of Glazebrook JS, Campbell RSF (1987) Diseases of barramundi most remaining bamim'U~d!-liK~lY recovered suffi­ (Lates calcarifer) in Australia: a review. In: Copland JW, ciently to fight bff infectionsf' :However, previous Grey DL (eds) Management of wild and cultured sea bass/barramundi (Lates calcarifer): proceedings of an }lnconfirmed outbreak§·dfN; 'inel1e!ni pn L. calcarifer international workshop, Darwin, N.T. Australia, 24-30 that were likely mlsdiagnosed'asSfi'l::!JJfococcus infec­ September 1986. Australian Centre for International Agri­ tions at this farm occurred when seawater tempera­ cultural Research (ACIAR) Proceedings No. 20. ACIAR, tures were not excessively low. This ubiquitous cap­ Canberra, p 204-206 salid is probably able to infect weaker fish that are Humason GL (1979) tissue techniques, 4th edn. WH Freeman and Company, San Francisco compromised because of poor nutrition, sickness, re­ Jahn TL, Kuhn LR (1932) The life-history of Epibdella melleni duced efficiency or natural variation of the immune MacCallum, 1927, a monogenetic trematode parasitic on system irrespective of seawater temperature, which marine fishes. BioI Bull 62:89-111 makes it a further threat in marlculture. Kaneko JJ, Yamada R, Brock JA, Nakamura RM (1988) Infec­ tion of tilapia, Oreochromis mossambicus (Trewavas), by a & Glazebrook Campbell (1987) noted that the range marine monogenean, Neobenedenia melleni (MacCallum, of disease agents identified in Australian barramundi 1927) Yamaguti, 1963 in Kaneohe Bay, Hawaii, USA, and overlapped considerably with those reported from its treatment. J Fish Dis 11 :295-300 other countries. We suggest that Neobenedenia mel­ Koesharyani I, Zafran, Yuasa Y, Hatai K (1999) Two species of leni may also be a pathogen to barramundi and other capsalid monogeneans infecting cultured humpback grouper Cromileptes altivelis in Indonesia. Fish Patho134: fin-fish cultured in southeast Asia. Mariculture is in its 165-166 infancy in Australia, which, combined with a lack of Leong TS (1997) Control of parasites in cultured marine fin­ diagnostic knowledge, may be why no outbreaks of N. fishes in southeast Asia: an overview. Int J Parasitol 27: melleni have been reported until now. However, N. 1177-1184 MacCallum GA (1927) A new ectoparasitic trematode, Epib­ melleni has a broad geographic range and infects a della melleni, sp. nov. Zoopathologica 1:291-300 multitude of wild and cultured fish species. Taking into Mueller KW, Watanabe WO, Head WD (1992) Effect of salin­ account these 2 key factors alone, we predict that marl­ ity on hatching in Neobenedenia melleni, a monogenean culture of not only barramundi, but also other fin-fish ectoparasite of seawater-cultured tilapia. J World species in Australia, is under threat of infection by N. Aquacult Soc 23:199-204 Ogawa K, Yokoyama H (1998) Parasitic diseases of cultured melleni. We encourage the reporting of any disease marine fish in Japan. Fish Patho133:303-309 agents in mariculture to appropriate authorities and Ogawa K, Bondad-Reantaso M, Fukudome M, Wakabayashi experts so that pathogens are identified correctly, suit­ H (1995) Neobenedenia girellae (Hargis, 1955) Yamaguti, able records are maintained and voucher specimens 1963 (Monogenea: Capsalidae) from cultured marine fishes of Japan. J Parasito181:223-227 are deposited in recognised, curated museum collec­ Pritchard MH, Kruse GOW (1982) The collection and preser­ tions. These actions will provide an invaluable founda­ vation of animal parasites. University of Nebraska Press, tion of data for the future study of marine pathogens of Lincoln cultivated species. Rimmer M (1995) Barramundi farming an introduction. Department of Primary Industries, Queensland Roff JC, Hopcroft RR (1986) High precision microcomputer Acknowledgements. We thank the personnel from the barra­ based measuring system for ecological research. Can J mundi farm for providing us with specimens and for useful Fish Aquat Sci 43:2044-2048 information and discussions about their fluke outbreak. In Thoney DA, Hargis WJ Jr (1991) Monogenea (Platy­ particular, we are indebted to Mr Dave Borgeldt, Mr Terry helminthes) as hazards for fish in confinement. Annu Rev Colson, Mr Reg Geary and Mr Justin Goc for their time, inter­ Fish Dis 1:133-153 est and assistance. M.R.D. was in receipt of an Australian Whittington rD, Horton MA (1996) A revision of Neobenede­ Postgraduate Award during this investigation. This study was nia Yamaguti, 1963 (Monogenea: Capsalidae) including a funded in part by Australian Research Council grant no. redescription of N. melleni (MacCallum, 1927) Yamaguti, A19801424. 1963. J Nat Hist 30:1113-1156 Whittington rD, Deveney MR, Wyborn SJ (2001) A revision of LITERATURE CITED Benedenia Diesing, 1858 including a redescription of B. sciaenae (van Beneden, 1856) Odhner, 1905 and recogni­ Anderson IG (1996) Fish health: the role of stress and hus­ tion of Menziesia Gibson, 1976 (Monogenea: Capsalidae). bandry. In: Barlow CG, Curtis MC (eds) Australian barra­ J Nat Hist 35:663-777 mundi farming workshop 1996 proceedings. Department Yamaguti S (1968) Monogenetic trematodes of Hawaiian of Primary Industries. Freshwater Fisheries and Aquacul- fishes. University of Hawaii Press, Honolulu

Editorial responsibility: Wolfgflng Korting, Submitted: February 19, 2001; Accepted: June 10, 2001 Hannover, Germany Proofs received from author(s): July 25, 2001